Cloning, Expression And Site-directed Mutation Of GH Family 7 Cellulases From Chaetomium Thermophilum CT2

Chaetomium thermophile CT2 is an extremophile of high optimum growth temperature. The cellulose produced by this organism maintains excellent catalytic performance even at high temperature. This feature is favorable for wide industrial applications. This study obtained the cellobiohydrolase genes CBH3 and the endo-glucanase EG2 genes from C. thermophilum CT2 based on reverse transcription-polymerase chain reaction(RT-PCR), and analyzed the high level expression, purification properties of these two proteins using Pichia pastoris induced expression system; Five amino acid sites were mutated according to the homology modeling of the cellobiohydrolase genes CBH3. We also expressed and analyzed these five mutated protein respectively.CBH3 was expressed by strain WTCBH3, the molecular of this enzyme was 47 k Da, the optimum temperature and p H of the enzyme activity were 60? and 5.0, respectively. CBH3 remained 80% of its original activity after 1 hour at 70?, this result revealed the excellent thermostability of CBH3. CBH3 remained more than 90% activity in the temperature of 50?~60?, while when the temperature is above 65?, its activity decreased obviously as the temperature dropped; The molecular of EG2 was 45 k Da, the optimum temperature and p H of the enzyme activity were 55? and 5.0, respectively. EG2 remained more than 70% of its original activity after 1 hour at 70?.The sequence similarity between Cel7 B and CBH3 is 80.65%, and the spatial structure of Cel7 B was very clear, all the parameters indicated its possible to be a model of CBH3. To screen for the key amino acid sites which were related to the bingding of the substrate in CBH3, we adopted the Site-directed mutagenesis method, through which we switched 5 amino acids into alanine.The optimum temperature and p H of the enzyme activity of CBH3-246 Y, CBH3-250 R, CBH3-261 D, CBH3-337 D, CBH3-391 R were 60 ? and 5.0, respectively. The enzyme activities of 5 mutants were decresed in different degrees compared to the wide type strain CBH3. Among those, the activity of CBH3-391 R decreased by an estimated 60 percent and the activities of CBH3- 246 Y, CBH3-261 D, CBH3-337 D decreased by 25%. The enzyme activity of the 4 mutants declined to less than 30% of its original activity in 40? except CBH3-250 R.According to the experimental results, it can be inferred that the 246 rd Tyr, 261 rd Asp, 337 Asp, 391 rd Arg in the sequence of CBH3 are all important amino acid residues related to substrate binding in the process of catalytic reaction. The 391 rd Arg is particularly important to the catalytic activity of CBH3.